Chronic myelogenous leukemia (CML) is a clonal disorder arising from the neoplastic transformation of hematopoietic stem cells. Bcr/Abl, the gene product of the characteristic (9;22) translocation (Philadelphia chromosome) functions as constitutive tyrosine kinase. Recently, the tyrosine kinase inhibitor Gleevec (Imatinib, STI571) was introduced as effective therapy for suppression of CML. While STI571 induces a high rate of complete hematological and cytogenetic remissions, residual leukemic cells are readily detected in the majority of patients and development of resistance to STI571 has been reported. Systemically administered interferon-alpha (INFalpha) had been the treatment of choice for CML prior to STI571. However, due to its short half-life, very high doses were required for therapeutic responses associated with undesirable systemic side effects. Clinical trials are ongoing combining both treatment modalities, STI571 and INFalpha. We hypothesized that the production of INFalpha and other biologically active proteins, by bone marrow resident cells might exert biological activity while decreasing systemic side effects. Mesenchymal stem cells (MSC) are non-hematopoietic cells residing in the bone marrow that can be isolated, expanded ex vivo, and transplanted. They "home back" to their site of origin, the bone marrow. We have extensive evidence that i.v. injected MSC contribute and proliferate in the marrow and also in the stroma of solid tumors and their metastases. We here propose to use MSC to produce regulated therapeutic genes in situ, including INFalpha, MDA7/CD24 and MRP2. Philadelphia chromosome (Ph')-negative MSC from both normal and CML donors were expanded ex vivo, and infected with an adeno-associated virus (AAV) expressing either CMV-driven INFalpha or progesterone regulated INFalpha. We compared this MSC-produced INFalpha with pharmacy grade INFalpha in vitro and in vivo. Both INFalpha's appeared identical in their ability to inhibit human Ph'-positive leukemias in vitro, but in the KBM5 model of CML blast crisis systemically injected IFNalpha was ineffective in vivo, whereas IFNalpha-MSC exerted significant anti-tumor effects and doubled the survival of tumor bearing animals. Exposure to INFalpha-MSC resulted in upregulation of MHC Class 1 antigens on CML patient cells. We are proposing to extend our preliminary in vitro and in vivo studies with the aim of developing a clinical trial of INFalpha-MSC in CML patients resistant to STI571 in chronic, accelerated and blast phases of CML, and relapsing after BMT. Furthermore, we propose to explore MSC-produced human melanoma differentiation-associated gene 7 (MDA7/IL24), a novel cancer growth suppressing and apoptosis-inducing member of the IL-10 family of cytokines, for the treatment of CML. MDA7 possesses Th1 cytokine activity and induces apoptosis in 60 tested cancers, but not in normal cells, independent of p53 status, but not in normal cells. It also exerts strong anti-angiogenic effects. We have evidence that MDA7-MSC inhibit preferentially the growth of STI571-resistant CML cells. Alternatively, we will investigate effects of MSC-produced MRP2, a MIP1 alpha-related protein, which is induced in CML by STI571. In conclusion, we propose to develop MSC as a platform for the in situ production of therapeutic proteins in CML.